Gold/Silica biochips: applications to Surface Plasmon Resonance and fluorescence quenching

We report Gold/Silica biochips for low cost biosensor devices. Firstly, the study of biochemical interactions on silica by means of Surface Plasmon Resonance (SPR) is presented. Secondly, Gold/Silica biochips are employed to reduce the strong quenching that occurs when a fluorophore is close to the gold surface. Furthermore, the control of the Silica-like thickness allows optimizing the distance between the metallic surface and the fluorophore in order to enhance the fluorescent signal. These results represent the first steps towards highly sensitive, specific and low cost biosensors based, for example, on Surface Plasmon Coupled Emission (SPCE) techniques

Antibody response against Trichinella spiralis in experimentally infected rats is dose dependent

Domestic pigs are the main representatives of the domestic cycle of Trichinella spiralis that play a role in transmission to humans. In Europe, backyard pigs of small household farms are the most important risks for humans to obtain trichinellosis. Rats might play a role in the transmission of Trichinella spiralis from domestic to sylvatic animals and vice versa. In order to be able to investigate the role of wild rats in the epidemiology of T. spiralis in The Netherlands, we studied the dynamics of antibody response after T. spiralis infections in experimental rats, using infection doses ranging from very low (10 muscle larvae, ML, per rat) to very high (16 000 ML per rat). To evaluate the feasibility of rats surviving high infection doses with T. spiralis, clinical and pathological parameters were quantified. Serological tools for detecting T. spiralis in rats were developed to quantitatively study the correlation between parasite load and immunological response. The results show that an infection dose-dependent antibody response was developed in rats after infection with as low as 10 ML up to a level of 10 000 ML. A positive correlation was found between the number of recovered ML and serum antibody levels, although specific measured antibody levels correspond to a wide range of LPG values. Serum antibodies of rats that were infected even with 10 or 25 ML could readily be detected by use of the T. spiralis western blot 2 weeks post infection. We conclude that based on these low infection doses, serologic tests are a useful tool to survey T. spiralis in wild rats

Pseudomonas aeruginosa Expresses a Functional Human Natriuretic Peptide Receptor Ortholog: Involvement in Biofilm Formation

This is the final version of the article. Available from the publisher via the DOI in this record.Considerable evidence exists that bacteria detect eukaryotic communication molecules and modify their virulence accordingly. In previous studies, it has been demonstrated that the increasingly antibiotic-resistant pathogen Pseudomonas aeruginosa can detect the human hormones brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) at micromolar concentrations. In response, the bacterium modifies its behavior to adapt to the host physiology, increasing its overall virulence. The possibility of identifying the bacterial sensor for these hormones and interfering with this sensing mechanism offers an exciting opportunity to directly affect the infection process. Here, we show that BNP and CNP strongly decrease P. aeruginosa biofilm formation. Isatin, an antagonist of human natriuretic peptide receptors (NPR), prevents this effect. Furthermore, the human NPR-C receptor agonist cANF(4-23) mimics the effects of natriuretic peptides on P. aeruginosa, while sANP, the NPR-A receptor agonist, appears to be weakly active. We show in silico that NPR-C, a preferential CNP receptor, and the P. aeruginosa protein AmiC have similar three-dimensional (3D) structures and that both CNP and isatin bind to AmiC. We demonstrate that CNP acts as an AmiC agonist, enhancing the expression of the ami operon in P. aeruginosa. Binding of CNP and NPR-C agonists to AmiC was confirmed by microscale thermophoresis. Finally, using an amiC mutant strain, we demonstrated that AmiC is essential for CNP effects on biofilm formation. In conclusion, the AmiC bacterial sensor possesses structural and pharmacological profiles similar to those of the human NPR-C receptor and appears to be a bacterial receptor for human hormones that enables P. aeruginosa to modulate biofilm expression. IMPORTANCE: The bacterium Pseudomonas aeruginosa is a highly dangerous opportunist pathogen for immunocompromised hosts, especially cystic fibrosis patients. The sites of P. aeruginosa infection are varied, with predominance in the human lung, in which bacteria are in contact with host molecular messengers such as hormones. The C-type natriuretic peptide (CNP), a hormone produced by lung cells, has been described as a bacterial virulence enhancer. In this study, we showed that the CNP hormone counteracts P. aeruginosa biofilm formation and we identified the bacterial protein AmiC as the sensor involved in the CNP effects. We showed that AmiC could bind specifically CNP. These results show for the first time that a human hormone could be sensed by bacteria through a specific protein, which is an ortholog of the human receptor NPR-C. The bacterium would be able to modify its lifestyle by favoring virulence factor production while reducing biofilm formation.We thank Magalie Barreau and Olivier Maillot for technical assistance. We thank Christine Farmer for linguistic insight for the manuscript. T. Rosay is a recipient of a doctoral fellowship from the French Ministry of Research (MRE). This work was supported by grants from the Communauté d’Agglomération d’Evreux, the Conseil Général de l’Eure, European Union (FEDER no. 31970), the French Association “Vaincre la Mucoviscidose” and the InterReg IVA PeReNE project

Blockchain and Government Transformation

In today’s digital age, transactions are made increasingly in the virtual universe. Some do all financial transactions online, but some do not even want to think about this possibility since the consideration is unreliable. To make online financial transactions more reliable, many technologies have already been tested. Since passwords, tokens, physical code generation accessories, there have been many attempts, being the most elaborate, the blockchain. Governments, especially in developed countries, often find it difficult to gain the trust of their citizens, especially when it comes to evidence of service provision and improvement of existing services. In developing countries, blockchain requests from governments would be useful in eliminating some important problems, such as corruption, while ensuring more effective deployment and distribution of resources. The adoption of such technologies can also help to facilitate better use of resources. In this paper, we will analyze the impact of the application of blockchain technology on e-government.2020-2

Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018): A Position Statement of the International Society for Extracellular Vesicles and Update of the MISEV2014 Guidelines

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points

Renal tubular Sirt1 attenuates diabetic albuminuria by epigenetically suppressing Claudin-1 overexpression in podocytes

Sirtuin 1 (Sirt1), a NAD[superscript +]-regulated deacetylase with numerous known positive effects on cellular and whole-body metabolism, is expressed in the renal cortex and medulla. It is known to have protective effects against age-related disease, including diabetes. Here we investigated the protective role of Sirt1 in diabetic renal damage. We found that Sirt1 in proximal tubules (PTs) was downregulated before albuminuria occurred in streptozotocin-induced or obese (db/db) diabetic mice. PT-specific SIRT1 transgenic and Sirt1 knockout mice showed prevention and aggravation of the glomerular changes that occur in diabetes, respectively, and nondiabetic knockout mice exhibited albuminuria, suggesting that Sirt1 in PTs affects glomerular function. Downregulation of Sirt1 and upregulation of the tight junction protein Claudin-1 by SIRT1-mediated epigenetic regulation in podocytes contributed to albuminuria. We did not observe these phenomena in 5/6 nephrectomized mice. We also demonstrated retrograde interplay from PTs to glomeruli using nicotinamide mononucleotide (NMN) from conditioned medium, measurement of the autofluorescence of photoactivatable NMN and injection of fluorescence-labeled NMN. In human subjects with diabetes, the levels of SIRT1 and Claudin-1 were correlated with proteinuria levels. These results suggest that Sirt1 in PTs protects against albuminuria in diabetes by maintaining NMN concentrations around glomeruli, thus influencing podocyte function.Japan. Ministry of Education, Culture, Sports, Science and Technology (Grant 22790800

Characterization of LINE-1 Ribonucleoprotein Particles

The average human genome contains a small cohort of active L1 retrotransposons that encode two proteins (ORF1p and ORF2p) required for their mobility (i.e., retrotransposition). Prior studies demonstrated that human ORF1p, L1 RNA, and an ORF2p-encoded reverse transcriptase activity are present in ribonucleoprotein (RNP) complexes. However, the inability to physically detect ORF2p from engineered human L1 constructs has remained a technical challenge in the field. Here, we have employed an epitope/RNA tagging strategy with engineered human L1 retrotransposons to identify ORF1p, ORF2p, and L1 RNA in a RNP complex. We next used this system to assess how mutations in ORF1p and/or ORF2p impact RNP formation. Importantly, we demonstrate that mutations in the coiled-coil domain and RNA recognition motif of ORF1p, as well as the cysteine-rich domain of ORF2p, reduce the levels of ORF1p and/or ORF2p in L1 RNPs. Finally, we used this tagging strategy to localize the L1–encoded proteins and L1 RNA to cytoplasmic foci that often were associated with stress granules. Thus, we conclude that a precise interplay among ORF1p, ORF2p, and L1 RNA is critical for L1 RNP assembly, function, and L1 retrotransposition

Integrated analysis of DNA methylation and gene expression reveals specific signaling pathways associated with platinum resistance in ovarian cancer

<p>Abstract</p> <p>Background</p> <p>Cisplatin and carboplatin are the primary first-line therapies for the treatment of ovarian cancer. However, resistance to these platinum-based drugs occurs in the large majority of initially responsive tumors, resulting in fully chemoresistant, fatal disease. Although the precise mechanism(s) underlying the development of platinum resistance in late-stage ovarian cancer patients currently remains unknown, CpG-island (CGI) methylation, a phenomenon strongly associated with aberrant gene silencing and ovarian tumorigenesis, may contribute to this devastating condition.</p> <p>Methods</p> <p>To model the onset of drug resistance, and investigate DNA methylation and gene expression alterations associated with platinum resistance, we treated clonally derived, drug-sensitive A2780 epithelial ovarian cancer cells with increasing concentrations of cisplatin. After several cycles of drug selection, the isogenic drug-sensitive and -resistant pairs were subjected to global CGI methylation and mRNA expression microarray analyses. To identify chemoresistance-associated, biological pathways likely impacted by DNA methylation, promoter CGI methylation and mRNA expression profiles were integrated and subjected to pathway enrichment analysis.</p> <p>Results</p> <p>Promoter CGI methylation revealed a positive association (Spearman correlation of 0.99) between the total number of hypermethylated CGIs and GI₅₀values (<it>i.e</it>., increased drug resistance) following successive cisplatin treatment cycles. In accord with that result, chemoresistance was reversible by DNA methylation inhibitors. Pathway enrichment analysis revealed hypermethylation-mediated repression of cell adhesion and tight junction pathways and hypomethylation-mediated activation of the cell growth-promoting pathways PI3K/Akt, TGF-beta, and cell cycle progression, which may contribute to the onset of chemoresistance in ovarian cancer cells.</p> <p>Conclusion</p> <p>Selective epigenetic disruption of distinct biological pathways was observed during development of platinum resistance in ovarian cancer. Integrated analysis of DNA methylation and gene expression may allow for the identification of new therapeutic targets and/or biomarkers prognostic of disease response. Finally, our results suggest that epigenetic therapies may facilitate the prevention or reversal of transcriptional repression responsible for chemoresistance and the restoration of sensitivity to platinum-based chemotherapeutics.</p

The In Vivo Kinetics of RNA Polymerase II Elongation during Co-Transcriptional Splicing

Kinetic analysis shows that RNA polymerase elongation kinetics are not modulated by co-transcriptional splicing and that post-transcriptional splicing can proceed at the site of transcription without the presence of the polymerase

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

© 2024 The Authors. Journal of Extracellular Vesicles, published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY), https://creativecommons.org/licenses/by/4.0/Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.Peer reviewe